Talk:'Round-the-horn site-directed mutagenesis

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I would not recommend using desalted primers for this procedure, even if they are relatively short (~20 bp each). I did so and got 1, 2, and 3 base pair deletions at the ligation site. I sequenced 10 constructs and they were all like this, presumably due to truncated primers. Maybe add a warning to use purified primers? This needs to be factored into the tradeoffs for this method. Quikchange works quite well with desalted primers in my hands, probably because the mutation is in the middle of the primer. --Jonathan Lake 12:19, 17 May 2010 (EDT)

We used to occasionally observe a one or two base deletion at the stitching site, only in one of several sequenced clones. I also suspected at the time the oligo quality (which should not show up at a specific place in the oligo because a synthesis deletion should be scattered randomly). I now suspect some activity of the enzyme used or an intra-cellular editing activity. We don't observe this artifact any more and have used it hundreds of times now with standard desalted oligos (Eurofins Operon). Perhaps it's because we only use fusion enzymes now (love NEB's Q5). Also, we almost exclusively clone into DH5-alpha, so maybe the host matters (nibbling the ends before ligation?). --Sean Moore 12:09, 26 February 2015 (EDT)

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